Interaction of model peptides with the peptidyl-prolyl cis/trans isomerases SurA and PpiD

Over the last 30 years there has been deep interest into the folding of proteins and the mechanisms in which they fold. One of the rate limiting steps of protein folding has been shown to be the cis/trans isomerisation of proline residues, which is catalysed by a range of peptidyl prolyl cis/trans isomerases (PPIases). In the periplasmic space of E.coli and other gram-negative bacteria two PPIases, SurA and PpiD, have been identified, which show high sequence similarity to the catalytic domain of the small PPIase parvulin. This observation raises the question about the biological significance of two apparently similar enzymes being present in the same cellular compartment: Do they interact with different substrates or do they catalyse different reactions? SurA has recently shown to be an important function in the formation of outer membrane porins and the pilus. Recent developments have revealed the crystallographic structure of SurA, indicating a possible region that could act as a binding cleft which corresponds to an area that has been shown to bind peptides. The substrate binding motif of PpiD has not been characterised so far and no biochemical data have been available on how this folding catalyst recognizes and interacts with substrates.

To characterise the interaction between model peptides and the periplasmic PPIases PpiD and SurA from E.coli, a chemical cross-linking strategy was employed that has been used previously to elucidate the interaction of substrates with PDI.

It was found that PpiD interacted with a range of model peptides independent of whether they contained proline residues or not. It was further demonstrate here that PpiD and SurA could interact with similar amino acids in a specific model peptide and therefore have overlapping substrate recognition motifs. However, the binding motif of PpiD is less specific than the one of SurA, indicating that the two PPIases might interact with different substrates, which was confirmed by in vivo crosslinking. We therefore propose that although PpiD and SurA have overlapping substrate recognition motifs they fulfil different functions in the cell. Molecular modeling coupled with mutagenesis has highlighted residues within SurA that are significant for the overall structure of the protein and subsequently the binding ability. This study has also shown that the N-terminal domain of SurA alone is sufficient for the binding

xx Abstract of peptides and does not require the C-terminal domain for binding but does require the C-terminal domain for stability.